Small antibody fragments show exciting promise for use as therapeutic agents, diagnostic reagents, and for biochemical research. Thus, they are needed in large amounts, and the expression of antibody fragments, e.g. Fv, single-chain Fv (scFv), or Fab in the periplasm of E. coli (Skerra & Plückthun 1988; Better et al., 1988) is now used routinely in many laboratories. Expression yields vary widely, however, especially in the case of scFvs. While some fragments yield up to several mg of functional, soluble protein per litre and OD of culture broth in shake flask culture (Carter et al., 1992, Plückthun et al. 1996), other fragments may almost exclusively lead to insoluble material, often found in so-called inclusion bodies. Functional protein may be obtained from the latter in modest yields by a laborious and time-consuming refolding process. The factors influencing antibody expression levels are still only poorly understood. Folding efficiency and stability of the antibody fragments, protease liability and toxicity of the expressed proteins to the host cells often severely limit actual production levels, and several attempts have been tried to increase expression yields. For example, Knappik & Plückthun (1995) have identified key residues in the antibody framework which influence expression yields dramatically. Similarly, Ullrich et al. (1995) found that point mutations in the CDRs can increase the yields in periplasmic antibody fragment expression. Nevertheless, these strategies are only applicable to a few antibodies.
The observations by Knappik & Plückthun (1995) indicate that optimising those parts of the antibody fragment which are not directly involved in antigen recognition can significantly improve folding properties and production yields of recombinant Fv and scFv constructs. The causes for the improved expression behaviour lie in the decreased aggregation behaviour of these molecules. For other molecules, fragment stability and protease resistance may also be affected. The understanding of how specific sequence modifications change these properties is still very limited and currently under active investigation.
Single-chain Fv fragments (scFvs) are recombinant antibody fragments consisting of the variable domains of the heavy and light chain; connected by a flexible peptide linker12 13. These fragments conserve the monovalent binding affinity and the specificity of the parent mAb and can be efficiently produced in bacteria14. ScFvs can be constructed by cloning the variable domains of a mAb showing interesting binding properties from hybridoma cells or by direct selection of scFv fragments with the desired specificity from immunized or naive phage libraries15 16. Frequently scFvs cloned from hybridomas show poor production yields and low thermodynamic stability which limit their usefulness for in vivo applications17, whereas scFvs selected from phage libraries have already undergone selection not only for antigen binding, but also for stability and folding properties in the scFv format18.
For therapeutic applications, human antibodies or antibody fragments are preferred to avoid an immune response e.g. against a murine antibody fragment derived from a monoclonal antibody (HAMA response). To solve that problem, human antibody fragments can be obtained by screening human antibody libraries (EP-A1 0 859 841; Vaughan et al., 1996). Another solution is to transplant the specificity of a non-human monoclonal antibody by grafting the CDR regions onto a human framework (EP-B1 0 239 400). In an improvement of said technique, humanized antibodies or antibody fragments with improved binding behavior can be produced by incorporating additional residues derived from said non-human antibody (EP-B1 0 451 216). In addition to achieving humanization, these techniques allow to “repair” scFv fragments with suboptimal stability and/or folding yield by grafting of the CDRs of a scFv fragment with the desired binding affinity and specificity onto the framework of a different, better behaved scFv, as was shown for the fluorescein binding antibody fragment 4-4-20 whose CDRs were grafted on the 4D5-framework, leading to a clear improvement of both expression yield and thermodynamic stability18. The 4D5 framework itself is an artificial framework resulting from the human consensus sequence and was used for the humanization of the anti-c-erbB2 (p185Her2-ECD) 4D5 mAb (Herceptin™)19. Later studies showed the above average thermodynamic stability of the 4D5 antibody fragment20, which correlates to the thermal stability of this molecule (Wörn and Plückthun, 1999) and is apparently of general importance for the in vivo application of scFvs.
The murine monoclonal antibody (mAb) MOC31 recognizes the 38 kDa transmembrane epithelial glycoprotein-21 (EGP-2; also known as GA733-2, Ep-CAM or KSA). EGP-2 is regarded as a suitable target antigen for tumor imaging and therapy, since it is highly overexpressed on a variety of human carcinomas and is not shed into the circulation. Several clinical trials with anti-EGP-2 mAbs such as 17-1A, KS1/4 and MOC312,3,4 demonstrated the potential of these antibodies for active and passive immunotherapy of human carcinomas. The exact function of the transmembrane glycoprotein EGP-2 is not yet known, although a role in cell-cell association has been proposed (Simon et al., 1990). Recent reports identify EGP-2 as a homophilic cell-cell adhesion molecule5,6 and EGP-2 has been identified as a potential modifier of invasiveness and chemoresponsiveness7. In a study evaluating the potential of new immunotherapeutics targeted to EGP-2, exotoxin-A (ETA) chemically fused to mAb MOC31 was found to retard the growth of large-tumors8.
Carcinoma-associated antigens such as c-erbB2 and EGF-receptor, as well as EGP-2 have served as targets for radiolabelled antibodies for tumor imaging and therapy. Effort have been made to improve the targeting efficiency by reducing the molecular weight and thereby increasing the tissue penetration and serum clearance of such antibody-based constructs.
Fab, (Fab)2, dsFv and scFv fragments, generated by recombinant antibody technology, have great potential in this respect9,10, although up to now the optimal formats concerning stability, molecular weight and affinity have not been determined and have to be fine-tuned for the different antibody-effector fusion proteins depending on the special in vivo system and application goal11.
For the development of new antibody fragment based imaging and therapeutic reagents directed to the pancarcinoma associated antigen epithelial glycoprotein-2 the variable domains of the murine anti-EGP-2 hybridoma MOC31 was cloned in the single-chain Fv fragment format16. Although the resulting scFv showed the expected binding affinity and specificity towards EGP-2, which was also shown on tissues sections in immunohistostaining experiments by others30, it was poorly expressed in the periplasm of bacteria. In vivo targeting experiments in nude mice employing this scFv fragment failed. The scFv not only did not accumulate in the tumor, but also showed slower clearance rates than an irrelevant control scFv directed against fluorescein. It could be shown that the MOC31 scFv formed high molecular weight aggregates and rapidly lost its activity when incubated in serum at body temperature (37° C.). This was primarily due to insufficient thermal stability rather than proteolytic degradation, since similar precipitation and loss of immunoactivity could also be observed upon incubation of highly purified scFv in PBS at 37° C.
To derive from this aggregation-prone and thermally instable scFv a molecule suitable for immunotherapeutic application, the biophysical properties of the construct had to be improved. Basically, two avenues were open to approach this goal: In-vitro evolution of the MOC31 scFv towards better thermal stability by combining randomization with selection for improved functionality35 at elevated temperature or the transfer of the binding specificity of the anti-EGP-2 scFv MOC31 onto a scFv framework with above average biophysical properties by CbR grafting18. Although the first option has been successfully used to achieve extremely stable scFvs35, the second option had the added advantage that by choosing a human framework sequence for the graft, a humanization could be achieved at the same time, thus reducing the potential immunogenicity of future immunotherapeutic reagents. It was therefore decided to graft the anti-EGP-2 scFv MOC31 binding specificity onto the artificial human consensus framework of scFv 4D5, essentially corresponding to the germline sequences IGVH 3-66 and IGVK 1-39 (IMGT). Grafting of complementary determining regions (CDRs) of mAbs for humanization has been used more than 100 times for humanization10 and can now be regarded as a standard technology. The 4D5 framework has been used successfully several times before as an CDR acceptor21,18,36.
This strategy proved successful, since the graft variant 4D5MOC-A showed binding characteristics indistinguishable to those of the parent antibody and scFv.
However, 4D5MOC-A showed only a thermal stability intermediate between that of the two parent molecules 4D5 and MOC31.
Biodistribution data indicated, that scFv MOC31, which lost most of its activity within less than 1 hour at 37° C. failed to enrich at the tumor, the graft variant 4D5MOC-A, stable for a few hours at 37° C. enriched only slightly.